Question

You have isolated a piece of DNA that you believe contains an interesting gene. Using a mix of RNA polymerase and ribonucleotides, you perform in vitro transcription. However, even though all of your controls work, no mRNA is created from your DNA fragment. Working backward, you note that your DNA preparation removed approximately 250 base pairs from the 5' end of the gene. The most likely explanation is that during DNA preparation_____

Answer 1

The most likely explanation for the failure of in vitro transcription after removing base pairs from the 5' end of the DNA is the unintentional deletion of the promoter region, which is essential for RNA polymerase binding and transcription initiation.

Step-by-step explanation:

Understanding in vitro Transcription Failure

During in vitro transcription, a specific sequence at the 5' end of the gene, typically known as the promoter region, is crucial for the binding of RNA polymerase and initiation of transcription. If approximately 250 base pairs were removed from the 5' end during DNA preparation, it is likely that the promoter region was either completely or partially deleted. Without this region, RNA polymerase would not be able to recognize where to begin transcription, hence no mRNA molecules could be formed from the isolated DNA fragment.

The process of transcription requires the DNA double helix to partially unwind to form a transcription bubble. RNA polymerase then synthesizes mRNA by reading the DNA template strand from 3' to 5', while the mRNA grows in a 5' to 3' direction. This process is terminated once a stop sequence is reached, releasing the new mRNA strand. If critical elements like the promoter are missing due to DNA preparation, transcription cannot commence, explaining the lack of mRNA synthesis observed.